Delayed rectifier potassium (K+) channels are responsible for shaping the action potential in all excitable cells, and control firing frequency in many cell types. K+ channels display an enormous range of functional diversity, due to subtle molecular differences and differential responsiveness to physiological modulators. The overall goal of our research is to understand the physiological and molecular mechanisms that underlie ion channel permeation and gating characteristics. In the widely distributed Kv2.1 potassium channel, a previously undescribed mechanism was discovered that underlies K+-dependent modulation of both permeation and gating functions of the channel. This mechanism, which involves a conformational change in the outer vestibule of the pore, is controlled by physiologically relevant changes in K+ concentration, and dramatically influences macroscopic current amplitude, activation rate, inactivation rate, and internal and external channel pharmacology. These changes are amplified in the presence of intracellular channel blockers, which include clinically used class III antiarrhythmics and local anesthetics. Preliminary data suggest that this same conformational change also underlies both K+- and pH-dependent modulation of currents in an important cardiac K+ channel (Kv1.5), which is a target for antiarrhythmics. We will use the patch clamp electrophysioloy technique, combined with molecular mutagenesis techniques, to understand the mechanisms by which K , and this K+- dependent change in channel conformation, modulate channel function. Specific aim one will examine the factors that control the K+-dependent change in outer vestibule conformation. Specific aim two will examine the mechanisms by which the K+-dependent conformational change modulates channel gating. These experiments will test several hypotheses regarding the mechanisms that link the channel pore to the gating process. Specific aim three will test the hypothesis that this same mechanism underlies the pH- and K+-dependent modulation of the Kv1.5 channel, and the more general hypothesis that this conformational change represents a general mechanism used by K+ channels to modulate current amplitude and gating properties. These experiments will lead to an understanding of how this novel mechanism modulates channel properties. Furthermore, these experiments will lead to a better understanding of how intracellular channel blockers interact with external pH and K+ to produce physiological and pathological consequences in both brain and heart.